Methods for the prevention or treatment of heart failure

ABSTRACT

The disclosure provides methods of preventing or treating heart failure in a mammalian subject. The methods comprise administering to the subject an effective amount of an aromatic-cationic peptide to subjects in need thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/035,577, filed Sep. 24, 2013, which is a continuation of U.S.application Ser. No. 12/897,325, filed Oct. 4, 2010, which claimspriority to U.S. Provisional Application No. 61/248,681, filed Oct. 5,2009, and U.S. Provisional Application No. 61/289,483, filed Dec. 23,2009, the entire contents of all of which are hereby incorporated byreference in their entirety.

GOVERNMENT SUPPORT

This invention was made with United States government support awarded bythe following agency: NIH R01 HL101186, P30 AG013280, and P01 AG001751.The United States government has certain rights in this invention.

TECHNICAL FIELD

The present technology relates generally to compositions and methods ofpreventing or treating heart failure. In particular, the presenttechnology relates to administering aromatic-cationic peptides ineffective amounts to prevent or treat heart failure in mammaliansubjects.

BACKGROUND

The following description is provided to assist the understanding of thereader. None of the information provided or references cited is admittedto be prior art to the present invention.

Heart failure is a leading cause of mortality and morbidity worldwide.In the United States, it affects nearly 5 million people and is the onlymajor cardiovascular disorder on the rise. It is estimated that 400,000to 700,000 new cases of heart failure are diagnosed each year in theU.S. and the number of deaths in the U.S. attributable to this conditionhas more than doubled since 1979, currently averaging 250,000 annually.Although heart failure affects people of all ages, the risk of heartfailure increases with age and is most common among older people.Accordingly, the number of people living with heart failure is expectedto increase significantly as the elderly population grows over the nextfew decades. The causes of heart failure have been linked to variousdisorders including coronary artery disease, past myocardial infarction,hypertension, abnormal heart valves, cardiomyopathy or myocarditis,congenital heart disease, severe lung disease, diabetes, severe anemia,hyperthyroidism, arrhythmia or dysrhythmia.

Heart failure (HF), also called congestive heart failure, is commonlycharacterized by decreased cardiac output, decreased cardiaccontractility, abnormal diastolic compliance, reduced stroke volume, andpulmonary congestion. The clinical manifestations of heart failurereflect a decrease in the myocardial contractile state and a reductionin cardiac output. Apart from deficiencies in cardiac contractility, theHF disease state may arise from left ventricular failure, rightventricular failure, biventricular failure, systolic dysfunction,diastolic dysfunction, and pulmonary effects. A progressive decrease inthe contractile function of cardiac muscle, associated with heartdisease, often leads to hypoperfusion of critical organs.

SUMMARY

The present technology relates generally to the treatment or preventionof heart failure in mammals through administration of therapeuticallyeffective amounts of aromatic-cationic peptides to subjects in needthereof. In particular embodiments, the aromatic-cationic peptides treator prevent heart failure by enhancing mitochondrial function in cardiactissues.

In one aspect, the disclosure provides a method of treating orpreventing heart failure or hypertensive cardiomyopathy, comprisingadministering to said mammalian subject a therapeutically effectiveamount of an aromatic-cationic peptide. In some embodiments, thearomatic-cationic peptide is a peptide having:

at least one net positive charge;

a minimum of four amino acids;

a maximum of about twenty amino acids;

a relationship between the minimum number of net positive charges(p_(m)) and the total number of amino acid residues (r) wherein 3p_(m)is the largest number that is less than or equal to r+1; and arelationship between the minimum number of aromatic groups (a) and thetotal number of net positive charges (p_(t)) wherein 2a is the largestnumber that is less than or equal to p_(t)+1, except that when a is 1,p_(t) may also be 1. In particular embodiments, the mammalian subject isa human.

In one embodiment, 2p_(m) is the largest number that is less than orequal to r+1, and a may be equal to p_(t). The aromatic-cationic peptidemay be a water-soluble peptide having a minimum of two or a minimum ofthree positive charges.

In one embodiment, the peptide comprises one or more non-naturallyoccurring amino acids, for example, one or more D-amino acids. In someembodiments, the C-terminal carboxyl group of the amino acid at theC-terminus is amidated. In certain embodiments, the peptide has aminimum of four amino acids. The peptide may have a maximum of about 6,a maximum of about 9, or a maximum of about 12 amino acids.

In one embodiment, the peptide comprises a tyrosine or a2′,6′-dimethyltyrosine (Dmt) residue at the N-terminus. For example, thepeptide may have the formula Tyr-D-Arg-Phe-Lys-NH₂ (SS-01) or2′,6′-Dmt-D-Arg-Phe-Lys-NH₂ (SS-02). In another embodiment, the peptidecomprises a phenylalanine or a 2′,6′-dimethylphenylalanine residue atthe N-terminus. For example, the peptide may have the formulaPhe-D-Arg-Phe-Lys-NH₂ (SS-20) or 2′,6′-Dmp-D-Arg-Phe-Lys-NH₂. In aparticular embodiment, the aromatic-cationic peptide has the formulaD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ (referred to interchangeably as SS-31,MTP-131, or Bendavia™).

In one embodiment, the peptide is defined by formula I:

wherein R¹ and R² are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

R³ and R⁴ are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo; R⁵, R⁶, R⁷, R⁸, and R⁹ are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo; and n is an integer from 1 to 5.

In a particular embodiment, R¹ and R² are hydrogen; R³ and R⁴ aremethyl; R⁵, R⁶, R⁷, R⁸, and R⁹ are all hydrogen; and n is 4.

In one embodiment, the peptide is defined by formula II:

wherein R¹ and R² are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are each independentlyselected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo; and n is an integer from 1 to 5.

In a particular embodiment, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R11and R¹² are all hydrogen; and n is 4. In another embodiment, R¹, R², R³,R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹¹ are all hydrogen; R⁸ and R¹² are methyl;R¹⁰ is hydroxyl; and n is 4.

In one embodiment, the subject is suffering from heart failure. In oneembodiment, the heart failure results from hypertension; ischemic heartdisease; exposure to a cardiotoxic compound; myocarditis; thyroiddisease; viral infection; gingivitis; drug abuse; alcohol abuse;pericarditis; atherosclerosis; vascular disease; hypertrophiccardiomyopathy; acute myocardial infarction; left ventricular systolicdysfunction; coronary bypass surgery; starvation; an eating disorder; ora genetic defect. In one embodiment, the subject is sufferinghypertensive cardiomyopathy.

In one embodiment, myocardial contractility and cardiac output in thesubject administered the peptide are increased compared to a controlsubject not administered the peptide. In one embodiment, the myocardialcontractility and cardiac output in the subject are increased at least10% compared to a control subject not administered the peptide.

In one embodiment, the method further comprises separately, sequentiallyor simultaneously administering a cardiovascular agent to the subject.In one embodiment, the cardiovascular agent is selected from the groupconsisting of: an anti-arrhthymia agent, a vasodilator, an anti-anginalagent, a corticosteroid, a cardioglycoside, a diuretic, a sedative, anangiotensin converting enzyme (ACE) inhibitor, an angiotensin IIantagonist, a thrombolytic agent, a calcium channel blocker, athroboxane receptor antagonist, a radical scavenger, an anti-plateletdrug, a β-adrenaline receptor blocking drug, α-receptor blocking drug, asympathetic nerve inhibitor, a digitalis formulation, an inotrope, andan antihyperlipidemic drug.

In another aspect, the disclosure provides a method for increasingmyocardial contractility and cardiac output in a subject suffering fromheart failure or hypertensive cardiomyopathy comprising administering tothe subject a therapeutically effective amount of the peptideD-Arg-2′6′-Dmt-Lys-Phe-NH₂ or Phe-D-Arg-Phe-Lys-NH₂.

The aromatic-cationic peptides may be administered in a variety of ways.In some embodiments, the peptides may be administered orally, topically,intranasally, intraperitoneally, intravenously, subcutaneously, ortransdermally (e.g., by iontophoresis).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph of flow cytometric analysis of neonatal cardiomyocytesstimulated with Ang 11 (1 μM) and loaded with Mitosox (5 μM), anindicator of mitochondrial ROS.

FIGS. 2A and 2B are a series of charts showing the effects of SS-31 onblood pressure after a pressor dose of Ang II. FIG. 2A: Representativeblood pressure tracings of mice at baseline and after Ang II (1.1mg/kg/d) administered with a subcutaneous pump. FIG. 2B: Ang IIsignificantly increased systolic blood pressure by 27.2 mm Hg anddiastolic pressure by 24.8 mm Hg.

FIGS. 3A-3D are a series of charts showing that SS-31 amelioratesAng-II-induced cardiac hypertrophy and diastolic dysfunction. FIG. 3A:Ang II (1.1 mg/kg/d) for 4 weeks substantially increased LVMI in WTcontrol mice. Simultaneous administration of SS-31 (3 mg/kg/d)significantly attenuated the Ang II-induced increase in LVMI (leftpanel), to a similar extent as that observed in mice with inducibleoverexpression of mitochondrial catalase (i-mCAT, right panel). FIG. 3Band FIG. 3C: Left ventricular end-diastolic diameter (LVEDD) andfractional shortening (FS,%) were not significantly changed after 4weeks of Ang II in the presence or absence of mitochondrialantioxidants. FIG. 3D: Diastolic function measured by tissue Dopplerimaging of Ea/Aa significantly reduced after 4 weeks of Ang II, but thisis significantly ameliorated by SS-31 or genetic overexpression of mCAT.

FIGS. 4A-4E are a series of charts showing SS-31 attenuates Ang-IIinduced cardiac hypertrophy and fibrosis. FIG. 4A: Ang II significantlyincreased heart weight (normalized to tibia length) and this wassignificantly attenuated by SS-31. FIG. 4B: Quantitative PCR showed adramatic increase in atrial natriuretic peptide (ANP) gene expression,which was significantly prevented by SS-31. FIG. 4C: Representativehistopathology shows substantial perivascular fibrosis (PVF) andinterstitial fibrosis (IF) after Ang II, which was better protected inSS-31 treated hearts. FIG. 4D: Quantitative analysis of blue trichromestaining demonstrated a significant increase in ventricular fibrosisafter Ang II, and this was substantially attenuated by SS-31. FIG. 4E:Quantitative PCR showed upregulation of pro-collagen 1a2 mRNA after AngII, which was significantly reduced in SS-31 hearts.

FIGS. 5A and 5B are a series of charts showing mitochondrial proteincarbonyl and signaling for mitochondrial biogenesis increased after 4weeks of Ang II treatment, which was prevented by SS-31. FIG. 5A: Ang IIfor 4 weeks significantly increased cardiac mitochondrial proteincarbonyl content, an indicator of protein oxidative damage, and this wassignificantly ameliorated by SS-31. FIG. 5B: Quantitative PCR revealedsignificant upregulation of genes in mitochondrial biogenesis, all ofwhich were attenuated by SS-31. *p<0.05 compared with saline group,#p<0.05 compared with Ang II treated group.

FIGS. 6A-6C are a series of charts showing SS-31 acts downstream ofNADPH oxidase and reduces activation of p38 MAPK and apoptosis inresponse to Ang II. FIG. 6A: NADPH oxidase activity was significantlyincreased after Ang II. No significant effect of SS-31 was observed.FIG. 6B: Ang II for 4 weeks substantially induced apoptosis, as shown byincrease in cleaved (activated) caspase 3 and this was significantlyattenuated by SS-31. FIG. 6C: Phosphorylation of p38 MAP kinasesignificantly increased after Ang II, which was substantially lower inSS-31 treated hearts (upper panel). Protein levels of p38 MAP kinasealso increased after Ang II.

FIGS. 7A-7E are a series of charts showing SS-31 ameliorated cardiachypertrophy and failure in Gαq overexpressing mice. Echocardiography ofGαq mice with or without SS-31 treatment and WT littermates at 16 weeksof age. FIG. 7A: SS-31 (3 mg/kg/d) for 4 weeks (from age 12 to 16 weeks)significantly ameliorated the decline in systolic function, as indicatedby FS, in Gαq overexpressing mice. FIG. 7B and FIG. 7C: Chamberenlargement and impairment of diastolic function in Gαq mice wereslightly attenuated by SS-31 with borderline significance, p=0.08 and0.06, respectively. FIG. 7D: Worsening of myocardial performance index(MPI) in Gαq mice was significantly ameliorated by SS-31. FIG. 7E: Anincrease in normalized heart weight in Gαq mice was substantiallyprotected by SS-31, while increased normalized lung weight displayed amodest effect from SS-31 with borderline significance (p=0.09).

FIG. 8 is a diagrammatic illustration of the proposed effect ofmitochondrial antioxidant SS-31 on Ang II and Gαq-inducedcardiomyopathy. Mitochondrial antioxidant SS-31 acts downstream toAngiotensin II receptor, Gαq, NADPH oxidase and upstream of p38 MAPK andapoptosis.

FIGS. 9A-9C are a series of charts showing both cardiac fibrosis andcardiac expression of Coll1 a2 gene were not significantly altered by 4weeks of SS-31 treatment (FIG. 9A and FIG. 9B). FIG. 9C shows cardiacmitochondrial protein carbonyl content significantly increased after AngII, which was reduced by SS-31.

DETAILED DESCRIPTION

It is to be appreciated that certain aspects, modes, embodiments,variations and features of the invention are described below in variouslevels of detail in order to provide a substantial understanding of thepresent invention. The definitions of certain terms as used in thisspecification are provided below. Unless defined otherwise, alltechnical and scientific terms used herein generally have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. For example, reference to “a cell” includesa combination of two or more cells, and the like.

As used herein, the “administration” of an agent, drug, or peptide to asubject includes any route of introducing or delivering to a subject acompound to perform its intended function. Administration can be carriedout by any suitable route, including orally, intranasally, parenterally(intravenously, intramuscularly, intraperitoneally, or subcutaneously),or topically. Administration includes self-administration and theadministration by another.

As used herein, the term “amino acid” includes naturally-occurring aminoacids and synthetic amino acids, as well as amino acid analogs and aminoacid mimetics that function in a manner similar to thenaturally-occurring amino acids. Naturally-occurring amino acids arethose encoded by the genetic code, as well as those amino acids that arelater modified, e.g., hydroxyproline, γ-carboxyglutamatc, andO-phosphoserine. Amino acid analogs refers to compounds that have thesame basic chemical structure as a naturally-occurring amino acid, i.e.,an α-carbon that is bound to a hydrogen, a carboxyl group, an aminogroup, and an R group, e.g., homoserine, norleucine, methioninesulfoxide, methionine methyl sulfonium. Such analogs have modified Rgroups (e.g., norleucine) or modified peptide backbones, but retain thesame basic chemical structure as a naturally-occurring amino acid. Aminoacid mimetics refers to chemical compounds that have a structure that isdifferent from the general chemical structure of an amino acid, but thatfunctions in a manner similar to a naturally-occurring amino acid. Aminoacids can be referred to herein by either their commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission.

As used herein, the term “effective amount” refers to a quantitysufficient to achieve a desired therapeutic and/or prophylactic effect,e.g., an amount which results in the prevention of or a decrease in,heart failure or one or more symptoms associated with heart failure. Inthe context of therapeutic or prophylactic applications, the amount of acomposition administered to the subject will depend on the type andseverity of the disease and on the characteristics of the individual,such as general health, age, sex, body weight and tolerance to drugs. Itwill also depend on the degree, severity and type of disease. Theskilled artisan will be able to determine appropriate dosages dependingon these and other factors. The compositions can also be administered incombination with one or more additional therapeutic compounds. In themethods described herein, the aromatic-cationic peptides may beadministered to a subject having one or more signs or symptoms of heartfailure, such as cardiomegaly, tachypnea, and hepatomegaly. For example,a “therapeutically effective amount” of the aromatic-cationic peptidesis meant levels in which the physiological effects of a heart failureare, at a minimum, ameliorated.

As used herein, the terms “congestive heart failure” (CHF), “chronicheart failure”, “acute heart failure”, and “heart failure” are usedinterchangeably, and refer to any condition characterized by abnormallylow cardiac output in which the heart is unable to pump blood at anadequate rate or in adequate volume. When the heart is unable toadequately pump blood to the rest of the body, or when one or more ofthe heart valves becomes stenotic or otherwise incompetent, blood canback up into the lungs, causing the lungs to become congested withfluid. If this backward flow occurs over an extended period of time,heart failure can result. Typical symptoms of heart failure includeshortness of breath (dyspnea), fatigue, weakness, difficulty breathingwhen lying flat, and swelling of the legs, ankles or abdomen (edema).Causes of heart failure are related to various disorders includingcoronary artery disease, systemic hypertension, cardiomyopathy ormyocarditis, congenital heart disease, abnormal heart valves or valvularheart disease, severe lung disease, diabetes, severe anemiahyperthyroidism, arrhythmia or dysrhythmia and myocardial infarction.The primary signs of congestive heart failure are: cardiomegaly(enlarged heart), tachypnea (rapid breathing; occurs in the case of leftside failure) and hepatomegaly (enlarged liver; occurs in the case ofright side failure).

As used herein, the term “hypertensive cardiomyopathy” refers to aweakened heart caused by the effects of hypertension (high bloodpressure). Over time, uncontrolled hypertension causes weakness of theheart muscle. As hypertensive cardiomyopathy worsens, it can lead tocongestive heart failure. Early symptoms of hypertensive cardiomyopathyinclude cough, weakness, and fatigue. Additional symptoms ofhypertensive cardiomyopathy include leg swelling, weight gain,difficulty breathing when lying flat, increasing shortness of breathwith activity, and waking in the middle of the night short of breath.

An “isolated” or “purified” polypeptide or peptide is substantially freeof cellular material or other contaminating polypeptides from the cellor tissue source from which the agent is derived, or substantially freefrom chemical precursors or other chemicals when chemically synthesized.For example, an isolated aromatic-cationic peptide would be free ofmaterials that would interfere with diagnostic or therapeutic uses ofthe agent. Such interfering materials may include enzymes, hormones andother proteinaceous and nonproteinaceous solutes.

As used herein, the terms “polypeptide”, “peptide”, and “protein” areused interchangeably herein to mean a polymer comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. Polypeptide refers to both short chains,commonly referred to as peptides, glycopeptides or oligomers, and tolonger chains, generally referred to as proteins. Polypeptides maycontain amino acids other than the 20 gene-encoded amino acids.Polypeptides include amino acid sequences modified either by naturalprocesses, such as post-translational processing, or by chemicalmodification techniques that are well known in the art.

As used herein, the term “simultaneous” therapeutic use refers to theadministration of at least two active ingredients by the same route andat the same time or at substantially the same time.

As used herein, the term “separate” therapeutic use refers to anadministration of at least two active ingredients at the same time or atsubstantially the same time by different routes.

As used herein, the term “sequential” therapeutic use refers toadministration of at least two active ingredients at different times,the administration route being identical or different. Moreparticularly, sequential use refers to the whole administration of oneof the active ingredients before administration of the other or otherscommences. It is thus possible to administer one of the activeingredients over several minutes, hours, or days before administeringthe other active ingredient or ingredients. There is no simultaneoustreatment in this case.

As used herein, the terms “treating” or “treatment” or “alleviation”refers to therapeutic treatment, wherein the object is to prevent orslow down (lessen) the targeted pathologic condition or disorder. Asubject is successfully “treated” for heart failure if, after receivinga therapeutic amount of the aromatic-cationic peptides according to themethods described herein, the subject shows observable and/or measurablereduction in or absence of one or more signs and symptoms of heartfailure, such as, e.g., cardiac output, myocardial contractile force,cardiomegaly, tachonea, and/or hepahemogaly. It is also to beappreciated that the various modes of treatment or prevention of medicalconditions as described are intended to mean “substantial”, whichincludes total but also less than total treatment or prevention, andwherein some biologically or medically relevant result is achieved.Treating heart failure, as used herein, also refers to treating any oneor more of the conditions underlying heart failure, including, withoutlimitation, decreased cardiac contractility, abnormal diastoliccompliance, reduced stroke volume, pulmonary congestion, and decreasedcardiac output.

As used herein, “prevention” or “preventing” of a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset or reduces theseverity of one or more symptoms of the disorder or condition relativeto the untreated control sample. As used herein, preventing heartfailure includes preventing the initiation of heart failure, delayingthe initiation of heart failure, preventing the progression oradvancement of heart failure, slowing the progression or advancement ofheart failure, delaying the progression or advancement of heart failure,and reversing the progression of heart failure from an advanced to aless advanced stage.

Aromatic-Cationic Peptides

The present technology relates to the treatment or prevention of heartfailure and related conditions by administration of certainaromatic-cationic peptides. The aromatic-cationic peptides arewater-soluble and highly polar. Despite these properties, the peptidescan readily penetrate cell membranes. The aromatic-cationic peptidestypically include a minimum of three amino acids or a minimum of fouramino acids, covalently joined by peptide bonds. The maximum number ofamino acids present in the aromatic-cationic peptides is about twentyamino acids covalently joined by peptide bonds. Suitably, the maximumnumber of amino acids is about twelve, more preferably about nine, andmost preferably about six.

The amino acids of the aromatic-cationic peptides can be any amino acid.As used herein, the term “amino acid” is used to refer to any organicmolecule that contains at least one amino group and at least onecarboxyl group. Typically, at least one amino group is at the α positionrelative to a carboxyl group. The amino acids may be naturallyoccurring. Naturally occurring amino acids include, for example, thetwenty most common levorotatory (L) amino acids normally found inmammalian proteins, i.e., alaninc (Ala), argininc (Arg), asparagine(Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gin), glutamicacid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine(Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline(Pro), serine (Ser), threonine (Thr), tryptophan, (Trp), tyrosine (Tyr),and valine (Val). Other naturally occurring amino acids include, forexample, amino acids that are synthesized in metabolic processes notassociated with protein synthesis. For example, the amino acidsornithine and citrulline are synthesized in mammalian metabolism duringthe production of urea. Another example of a naturally occurring aminoacid includes hydroxyproline (Hyp).

The peptides optionally contain one or more non-naturally occurringamino acids. Optimally, the peptide has no amino acids that arenaturally occurring. The non-naturally occurring amino acids may belevorotary (L-), dextrorotatory (D-), or mixtures thereof. Non-naturallyoccurring amino acids are those amino acids that typically are notsynthesized in normal metabolic processes in living organisms, and donot naturally occur in proteins. In addition, the non-naturallyoccurring amino acids suitably are also not recognized by commonproteases. The non-naturally occurring amino acid can be present at anyposition in the peptide. For example, the non-naturally occurring aminoacid can be at the N-terminus, the C-terminus, or at any positionbetween the N-terminus and the C-terminus.

The non-natural amino acids may, for example, comprise alkyl, aryl, oralkylaryl groups not found in natural amino acids. Some examples ofnon-natural alkyl amino acids include α-aminobutyric acid,β-aminobutyric acid, γ-aminobutyric acid, δ-aminovaleric acid, andε-aminocaproic acid. Some examples of non-natural aryl amino acidsinclude ortho-, meta, and para-aminobenzoic acid. Some examples ofnon-natural alkylaryl amino acids include ortho-, meta-, andpara-aminophenylacetic acid, and γ-phenyl-β-aminobutyric acid.Non-naturally occurring amino acids include derivatives of naturallyoccurring amino acids. The derivatives of naturally occurring aminoacids may, for example, include the addition of one or more chemicalgroups to the naturally occurring amino acid.

For example, one or more chemical groups can be added to one or more ofthe 2′, 3′, 4′, 5′, or 6′ position of the aromatic ring of aphenylalanine or tyrosine residue, or the 4′, 5′, 6′, or 7′ position ofthe benzo ring of a tryptophan residue. The group can be any chemicalgroup that can be added to an aromatic ring. Some examples of suchgroups include branched or unbranched C₁-C₄ alkyl, such as methyl,ethyl, n-propyl, isopropyl, butyl, isobutyl, or t-butyl, C₁-C₄ alkyloxy(i.e., alkoxy), amino, C₁-C₄ alkylamino and C₁-C₄ dialkylamino (e.g.,methylamino, dimethylamino), nitro, hydroxyl, halo (i.e., fluoro,chloro, bromo, or iodo). Some specific examples of non-naturallyoccurring derivatives of naturally occurring amino acids includenorvaline (Nva) and norleucine (Nle).

Another example of a modification of an amino acid in a peptide is thederivatization of a carboxyl group of an aspartic acid or a glutamicacid residue of the peptide. One example of derivatization is amidationwith ammonia or with a primary or secondary amine, e.g. methylamine,ethylamine, dimethylamine or diethylamine. Another example ofderivatization includes esterification with, for example, methyl orethyl alcohol. Another such modification includes derivatization of anamino group of a lysine, arginine, or histidine residue. For example,such amino groups can be acylated. Some suitable acyl groups include,for example, a benzoyl group or an alkanoyl group comprising any of theC₁-C₄ alkyl groups mentioned above, such as an acetyl or propionylgroup.

The non-naturally occurring amino acids are suitably resistant orinsensitive to common proteases. Examples of non-naturally occurringamino acids that are resistant or insensitive to proteases include thedextrorotatory (D-) form of any of the above-mentioned naturallyoccurring L-amino acids, as well as L- and/or D-non-naturally occurringamino acids. The D-amino acids do not normally occur in proteins,although they are found in certain peptide antibiotics that aresynthesized by means other than the normal ribosomal protein syntheticmachinery of the cell. As used herein, the D-amino acids are consideredto be non-naturally occurring amino acids.

In order to minimize protease sensitivity, the peptides should have lessthan five, preferably less than four, more preferably less than three,and most preferably, less than two contiguous L-amino acids recognizedby common proteases, irrespective of whether the amino acids arenaturally or non-naturally occurring. Optimally, the peptide has onlyD-amino acids, and no L-amino acids. If the peptide contains proteasesensitive sequences of amino acids, at least one of the amino acids ispreferably a non-naturally-occurring D-amino acid, thereby conferringprotease resistance. An example of a protease sensitive sequenceincludes two or more contiguous basic amino acids that are readilycleaved by common proteases, such as endopeptidases and trypsin.Examples of basic amino acids include arginine, lysine and histidine.

The aromatic-cationic peptides should have a minimum number of netpositive charges at physiological pH in comparison to the total numberof amino acid residues in the peptide. The minimum number of netpositive charges at physiological pH will be referred to below as(p_(m)). The total number of amino acid residues in the peptide will bereferred to below as (r). The minimum number of net positive chargesdiscussed below are all at physiological pH. The term “physiological pH”as used herein refers to the normal pH in the cells of the tissues andorgans of the mammalian body. For instance, the physiological pH of ahuman is normally approximately 7.4, but normal physiological pH inmammals may be any pH from about 7.0 to about 7.8.

“Net charge” as used herein refers to the balance of the number ofpositive charges and the number of negative charges carried by the aminoacids present in the peptide. In this specification, it is understoodthat net charges are measured at physiological pH. The naturallyoccurring amino acids that are positively charged at physiological pHinclude L-lysine, L-arginine, and L-histidine. The naturally occurringamino acids that are negatively charged at physiological pH includeL-aspartic acid and L-glutamic acid.

Typically, a peptide has a positively charged N-terminal amino group anda negatively charged C-terminal carboxyl group. The charges cancel eachother out at physiological pH. As an example of calculating net charge,the peptide Tyr-Arg-Phe-Lys-Glu-His-Trp-D-Arg has one negatively chargedamino acid (i.e., Glu) and four positively charged amino acids (i.e.,two Arg residues, one Lys, and one His). Therefore, the above peptidehas a net positive charge of three.

In one embodiment, the aromatic-cationic peptides have a relationshipbetween the minimum number of net positive charges at physiological pH(p_(m)) and the total number of amino acid residues (r) wherein 3p_(m)is the largest number that is less than or equal to r+1. In thisembodiment, the relationship between the minimum number of net positivecharges (p_(m)) and the total number of amino acid residues (r) is asfollows:

TABLE 1 Amino acid number and net positive charges (3p_(m) ≦ p + 1) (r)3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 (p_(m)) 1 1 2 2 2 3 3 3 44 4 5 5 5 6 6 6 7

In another embodiment, the aromatic-cationic peptides have arelationship between the minimum number of net positive charges (p_(m))and the total number of amino acid residues (r) wherein 2p_(m) is thelargest number that is less than or equal to r+1. In this embodiment,the relationship between the minimum number of net positive charges(p_(m)) and the total number of amino acid residues (r) is as follows:

TABLE 2 Amino acid number and net positive charges (2p_(m) ≦ p + 1) (r)3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 (p_(m)) 2 2 3 3 4 4 5 5 66 7 7 8 8 9 9 10 10

In one embodiment, the minimum number of net positive charges (p_(m))and the total number of amino acid residues (r) are equal. In anotherembodiment, the peptides have three or four amino acid residues and aminimum of one net positive charge, suitably, a minimum of two netpositive charges and more preferably a minimum of three net positivecharges.

It is also important that the aromatic-cationic peptides have a minimumnumber of aromatic groups in comparison to the total number of netpositive charges (m). The minimum number of aromatic groups will bereferred to below as (a). Naturally occurring amino acids that have anaromatic group include the amino acids histidine, tryptophan, tyrosine,and phenylalanine. For example, the hexapeptideLys-Gln-Tyr-D-Arg-Phe-Trp has a net positive charge of two (contributedby the lysine and arginine residues) and three aromatic groups(contributed by tyrosine, phenylalanine and tryptophan residues).

The aromatic-cationic peptides should also have a relationship betweenthe minimum number of aromatic groups (a) and the total number of netpositive charges at physiological pH (p_(t)) wherein 3a is the largestnumber that is less than or equal to p_(t)+1, except that when p_(t) is1, a may also be 1. In this embodiment, the relationship between theminimum number of aromatic groups (a) and the total number of netpositive charges (P_(t)) is as follows:

TABLE 3 Aromatic groups and net positive charges (3a ≦ p_(t) + 1 or a =p_(t) = 1) (p_(t)) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20(a) 1 1 1 1 2 2 2 3 3 3 4 4 4 5 5 5 6 6 6 7

In another embodiment, the aromatic-cationic peptides have arelationship between the minimum number of aromatic groups (a) and thetotal number of net positive charges (p_(t)) wherein 2a is the largestnumber that is less than or equal to p_(t)+1. In this embodiment, therelationship between the minimum number of aromatic amino acid residues(a) and the total number of net positive charges (p_(t)) is as follows:

TABLE 4 Aromatic groups and net positive charges (2a ≦ p_(t) + 1 or a =p_(t) = 1) (p_(t)) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20(a) 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10

In another embodiment, the number of aromatic groups (a) and the totalnumber of net positive charges (p_(t)) are equal.

Carboxyl groups, especially the terminal carboxyl group of a C-terminalamino acid, are suitably amidated with, for example, ammonia to form theC-terminal amide. Alternatively, the terminal carboxyl group of theC-terminal amino acid may be amidated with any primary or secondaryamine. The primary or secondary amine may, for example, be an alkyl,especially a branched or unbranched C₁-C₄ alkyl, or an aryl amine.Accordingly, the amino acid at the C-terminus of the peptide may beconverted to an amido, N-methylamido, N-ethylamido, N,N-dimethylamido,N,N-diethylamido, N-methyl-N-ethylamido, N-phenylamido orN-phenyl-N-ethylamido group. The free carboxylate groups of theasparagine, glutamine, aspartic acid, and glutamic acid residues notoccurring at the C-terminus of the aromatic-cationic peptides may alsobe amidated wherever they occur within the peptide. The amidation atthese internal positions may be with ammonia or any of the primary orsecondary amines described above.

In one embodiment, the aromatic-cationic peptide is a tripeptide havingtwo net positive charges and at least one aromatic amino acid. In aparticular embodiment, the aromatic-cationic peptide is a tripeptidehaving two net positive charges and two aromatic amino acids.

Aromatic-cationic peptides include, but are not limited to, thefollowing peptide examples:

Lys-D-Arg-Tyr-NH₂ Phe-D-Arg-His D-Tyr-Trp-Lys-NH₂ Trp-D-Lys-Tyr-Arg-NH₂Tyr-His-D-Gly-Met Phe-Arg-D-His-Asp Tyr-D-Arg-Phe-Lys-Glu-NH₂Met-Tyr-D-Lys-Phe-Arg D-His-Glu-Lys-Tyr-D-Phe-ArgLys-D-Gln-Tyr-Arg-D-Phe-Trp-NH₂ Phe-D-Arg-Lys-Trp-Tyr-D-Arg-HisGly-D-Phe-Lys-Tyr-His-D-Arg-Tyr-NH₂Val-D-Lys-His-Tyr-D-Phe-Ser-Tyr-Arg-NH₂Trp-Lys-Phe-D-Asp-Arg-Tyr-D-His-LysLys-Trp-D-Tyr-Arg-Asn-Phe-Tyr-D-His-NH₂Thr-Gly-Tyr-Arg-D-His-Phe-Trp-D-His-LysAsp-D-Trp-Lys-Tyr-D-His-Phe-Arg-D-Gly-Lys-NH₂D-His-Lys-Tyr-D-Phe-Glu-D-Asp-D-His-D-Lys-Arg- Trp-NH₂Ala-D-Phe-D-Arg-Tyr-Lys-D-Trp-His-D-Tyr-Gly-PheTyr-D-His-Phe-D-Arg-Asp-Lys-D-Arg-His-Trp-D- His-PhePhe-Phe-D-Tyr-Arg-Glu-Asp-D-Lys-Arg-D-Arg-His- Phe-NH₂Phe-Try-Lys-D-Arg-Trp-His-D-Lys-D-Lys-Glu-Arg-D- Tyr-ThrTyr-Asp-D-Lys-Tyr-Phe-D-Lys-D-Arg-Phe-Pro-D-Tyr- His-LysGlu-Arg-D-Lys-Tyr-D-Val-Phe-D-His-Trp-Arg-D-Gly- Tyr-Arg-D-Met-NH₂Arg-D-Leu-D-Tyr-Phe-Lys-Glu-D-Lys-Arg-D-Trp-Lys- D-Phe-Tyr-D-Arg-GlyD-Glu-Asp-Lys-D-Arg-D-His-Phe-Phe-D-Val-Tyr-Arg-Tyr-D-Tyr-Arg-His-Phe-NH₂Asp-Arg-D-Phe-Cys-Phe-D-Arg-D-Lys-Tyr-Arg-D-Tyr-Trp-D-His-Tyr-D-Phe-Lys-PheHis-Tyr-D-Arg-Trp-Lys-Phe-D-Asp-Ala-Arg-Cys-D-Tyr-His-Phe-D-Lys-Tyr-His-Ser-NH₂Gly-Ala-Lys-Phe-D-Lys-Glu-Arg-Tyr-His-D-Arg-D-Arg-Asp-Tyr-Trp-D-His-Trp-His-D-Lys-AspThr-Tyr-Arg-D-Lys-Trp-Tyr-Glu-Asp-D-Lys-D-Arg-His-Phe-D-Tyr-Gly-Val-Ile-D-His-Arg-Tyr-Lys-NH₂

In one embodiment, the peptides have mu-opioid receptor agonist activity(i.e., they activate the mu-opioid receptor). Peptides which havemu-opioid receptor agonist activity are typically those peptides whichhave a tyrosine residue or a tyrosine derivative at the N-terminus(i.e., the first amino acid position). Suitable derivatives of tyrosineinclude 2′-methyltyrosine (Mmt); 2′,6′-dimethyltyrosine (2′6′-Dmt);3′,5′-dimethyltyrosine (3′5′Dmt); N,2′,6′-trimethyltyrosine (Tmt); and2′-hydroxy-6′-methyltryosine (Hmt).

In one embodiment, a peptide that has mu-opioid receptor agonistactivity has the formula Tyr-D-Arg-Phe-Lys-NH₂ (referred to herein as“SS-01”). SS-01 has a net positive charge of three, contributed by theamino acids tyrosine, arginine, and lysine and has two aromatic groupscontributed by the amino acids phenylalanine and tyrosine. The tyrosineof SS-01 can be a modified derivative of tyrosine such as in2′,6′-dimethyltyrosine to produce the compound having the formula2′,6′-Dmt-D-Arg-Phe-Lys-NH₂ (referred to herein as “SS-02”). SS-02 has amolecular weight of 640 and carries a net three positive charge atphysiological pH. SS-02 readily penetrates the plasma membrane ofseveral mammalian cell types in an energy-independent manner (Zhao etal., J. Pharmacol Exp Ther., 304:425-432, 2003).

Alternatively, in other instances, the aromatic-cationic peptide doesnot have mu-opioid receptor agonist activity. For example, duringlong-term treatment, such as in a chronic disease state or condition,the use of an aromatic-cationic peptide that activates the mu-opioidreceptor may be contraindicated. In these instances, the potentiallyadverse or addictive effects of the aromatic-cationic peptide maypreclude the use of an aromatic-cationic peptide that activates themu-opioid receptor in the treatment regimen of a human patient or othermammal. Potential adverse effects may include sedation, constipation andrespiratory depression. In such instances an aromatic-cationic peptidethat does not activate the mu-opioid receptor may be an appropriatetreatment. Peptides that do not have mu-opioid receptor agonist activitygenerally do not have a tyrosine residue or a derivative of tyrosine atthe N-terminus (i.e., amino acid position 1). The amino acid at theN-terminus can be any naturally occurring or non-naturally occurringamino acid other than tyrosine. In one embodiment, the amino acid at theN-terminus is phenylalanine or its derivative. Exemplary derivatives ofphenylalanine include 2′-methylphenylalanine (Mmp),2′,6′-dimethylphenylalanine (2′,6′-Dmp), N,2′,6′-trimethylphenylalanine(Tmp), and 2′-hydroxy-6′-methylphenylalanine (Hmp).

An example of an aromatic-cationic peptide that does not have mu-opioidreceptor agonist activity has the formula Phe-D-Arg-Phe-Lys-NH₂(referred to herein as “SS-20”). Alternatively, the N-terminalphenylalanine can be a derivative of phenylalanine such as2′,6′-dimethylphenylalanine (2′6′-Dmp). SS-01 containing2′,6′-dimethylphenylalanine at amino acid position 1 has the formula2′,6′-Dmp-D-Arg-Phe-Lys-NH₂. In one embodiment, the amino acid sequenceof SS-02 is rearranged such that Dmt is not at the N-terminus. Anexample of such an aromatic-cationic peptide that does not havemu-opioid receptor agonist activity has the formulaD-Arg-2′6′-Dmt-Lys-Phe-NH₂.

Suitable substitution variants of the peptides listed herein includeconservative amino acid substitutions. Amino acids may be groupedaccording to their physicochemical characteristics as follows:

(a) Non-polar amino acids: Ala(A) Ser(S) Thr(T) Pro(P) Gly(G) Cys (C);

(b) Acidic amino acids: Asn(N) Asp(D) Glu(E) Gln(Q);

(c) Basic amino acids: His(H) Arg(R) Lys(K);

(d) Hydrophobic amino acids: Met(M) Leu(L) Ile(I) Val(V); and

(e) Aromatic amino acids: Phe(F) Tyr(Y) Trp(W) His (H).

Substitutions of an amino acid in a peptide by another amino acid in thesame group is referred to as a conservative substitution and maypreserve the physicochemical characteristics of the original peptide. Incontrast, substitutions of an amino acid in a peptide by another aminoacid in a different group is generally more likely to alter thecharacteristics of the original peptide.

Examples of peptides that activate mu-opioid receptors include, but arenot limited to, the aromatic-cationic peptides shown in Table 5.

TABLE 5 Peptide Analogs with Mu-Opioid Activity Amino Amino Amino AcidAcid Acid Amino Acid C-Terminal Position 1 Position 2 Position 3Position 4 Modification Tyr D-Arg Phe Lys NH₂ Tyr D-Arg Phc Orn NH₂ TyrD-Arg Phe Dab NH₂ Tyr D-Arg Phe Dap NH₂ 2′6′Dmt D-Arg Phe Lys NH₂2′6′Dmt D-Arg Phe Lys- NII₂ NII(CII₂)₂— NII-dns 2′6′Dmt D-Arg Phe Lys-NH₂ NH(CH₂)₂— NH-atn 2′6′Dmt D-Arg Phe dnsLys NH₂ 2′6′Dmt D-Cit Phe LysNH₂ 2′6′Dmt D-Cit Phe Ahp NH₂ 2′6′Dmt D-Arg Phe Orn NH₂ 2′6′Dmt D-ArgPhe Dab NH₂ 2′6′Dmt D-Arg Phe Dap NH₂ 2′6′Dmt D-Arg Phe Ahp(2- NH₂aminoheptanoic acid) Bio- D-Arg Phe Lys NH₂ 2′6′Dmt 3′5′Dmt D-Arg PheLys NH₂ 3′5′Dmt D-Arg Phe Orn NII₂ 3′5′Dmt D-Arg Phe Dab NH₂ 3′5′DmtD-Arg Phe Dap NH₂ Tyr D-Arg Tyr Lys NH₂ Tyr D-Arg Tyr Orn NH₂ Tyr D-ArgTyr Dab NH₂ Tyr D-Arg Tyr Dap NH₂ 2′6′Dmt D-Arg Tyr Lys NH₂ 2′6′DmtD-Arg Tyr Orn NH₂ 2′6′Dmt D-Arg Tyr Dab NH₂ 2′6′Dmt D-Arg Tyr Dap NH₂2′6′Dmt D-Arg 2′6′Dmt Lys NII₂ 2′6′Dmt D-Arg 2′6′Dmt Orn NH₂ 2′6′DmtD-Arg 2′6′Dmt Dab NH₂ 2′6′Dmt D-Arg 2′6′Dmt Dap NH₂ 3′5′Dmt D-Arg3′5′Dmt Arg NH₂ 3′5′Dmt D-Arg 3′5′Dmt Lys NH₂ 3′5′Dmt D-Arg 3′5′Dmt OrnNH₂ 3′5′Dmt D-Arg 3′5′Dmt Dab NH₂ Tyr D-Lys Phe Dap NH₂ Tyr D-Lys PheArg NH₂ Tyr D-Lys Phe Lys NH₂ Tyr D-Lys Phe Orn NH₂ 2′6′Dmt D-Lys PheDab NH₂ 2′6′Dmt D-Lys Phe Dap NH₂ 2′6′Dmt D-Lys Phe Arg NH₂ 2′6′DmtD-Lys Phe Lys NII₂ 3′5′Dmt D-Lys Phe Orn NH₂ 3′5′Dmt D-Lys Phe Dab NH₂3′5′Dmt D-Lys Phe Dap NH₂ 3′5′Dmt D-Lys Phe Arg NH₂ Tyr D-Lys Tyr LysNH₂ Tyr D-Lys Tyr Orn NH₂ Tyr D-Lys Tyr Dab NH₂ Tyr D-Lys Tyr Dap NH₂2′6′Dmt D-Lys Tyr Lys NH₂ 2′6′Dmt D-Lys Tyr Orn NH₂ 2′6′Dmt D-Lys TyrDab NH₂ 2′6′Dmt D-Lys Tyr Dap NH₂ 2′6′Dmt D-Lys 2′6′Dmt Lys NH₂ 2′6′DmtD-Lys 2′6′Dmt Orn NH₂ 2′6′Dmt D-Lys 2′6′Dmt Dab NH₂ 2′6′Dmt D-Lys2′6′Dmt Dap NH₂ 2′6′Dmt D-Arg Phe dnsDap NH₂ 2′6′Dmt D-Arg Phe atnDapNH₂ 3′5′Dmt D-Lys 3′5′Dmt Lys NH₂ 3′5′Dmt D-Lys 3′5′Dmt Orn NH₂ 3′5′DmtD-Lys 3′5′Dmt Dab NH₂ 3′5′Dmt D-Lys 3′5′Dmt Dap NII₂ Tyr D-Lys Phe ArgNH₂ Tyr D-Orn Phe Arg NH₂ Tyr D-Dab Phe Arg NH₂ Tyr D-Dap Phe Arg NH₂2′6′Dmt D-Arg Phe Arg NH₂ 2′6′Dmt D-Lys Phe Arg NH₂ 2′6′Dmt D-Orn PheArg NH₂ 2′6′Dmt D-Dab Phe Arg NH₂ 3′5′Dmt D-Dap Phe Arg NH₂ 3′5′DmtD-Arg Phe Arg NH₂ 3′5′Dmt D-Lys Phe Arg NII₂ 3′5′Dmt D-Orn Phe Arg NH₂Tyr D-Lys Tyr Arg NH₂ Tyr D-Orn Tyr Arg NH₂ Tyr D-Dab Tyr Arg NH₂ TyrD-Dap Tyr Arg NH₂ 2′6′Dmt D-Arg 2′6′Dmt Arg NH₂ 2′6′Dmt D-Lys 2′6′DmtArg NH₂ 2′6′Dmt D-Orn 2′6′Dmt Arg NH₂ 2′6′Dmt D-Dab 2′6′Dmt Arg NH₂3′5′Dmt D-Dap 3′5′Dmt Arg NH₂ 3′5′Dmt D-Arg 3′5′Dmt Arg NII₂ 3′5′DmtD-Lys 3′5′Dmt Arg NH₂ 3′5′Dmt D-Orn 3′5′Dmt Arg NH₂ Mmt D-Arg Phe LysNH₂ Mmt D-Arg Phe Orn NH₂ Mmt D-Arg Phe Dab NH₂ Mmt D-Arg Phe Dap NII₂Tmt D-Arg Phc Lys NH₂ Tmt D-Arg Phe Orn NH₂ Tmt D-Arg Phe Dab NH₂ TmtD-Arg Phe Dap NH₂ Hmt D-Arg Phe Lys NH₂ Hmt D-Arg Phe Orn NH₂ Hmt D-ArgPhe Dab NH₂ Hmt D-Arg Phe Dap NH₂ Mmt D-Lys Phe Lys NH₂ Mmt D-Lys PheOrn NH₂ Mmt D-Lys Phe Dab NH₂ Mmt D-Lys Phc Dap NH₂ Mmt D-Lys Phe ArgNH₂ Tmt D-Lys Phe Lys NH₂ Tmt D-Lys Phe Orn NH₂ Tmt D-Lys Phe Dab NH₂Tmt D-Lys Phe Dap NH₂ Tmt D-Lys Phe Arg NH₂ Hmt D-Lys Phe Lys NH₂ HmtD-Lys Phe Orn NH₂ Hmt D-Lys Phe Dab NH₂ Hmt D-Lys Phe Dap NII₂ Hmt D-LysPhc Arg NH₂ Mmt D-Lys Phe Arg NH₂ Mmt D-Orn Phe Arg NH₂ Mmt D-Dab PheArg NH₂ Mmt D-Dap Phe Arg NH₂ Mmt D-Arg Phe Arg NH₂ Tmt D-Lys Phe ArgNH₂ Tmt D-Orn Phe Arg NH₂ Tmt D-Dab Phe Arg NH₂ Tmt D-Dap Phe Arg NH₂Tmt D-Arg Phe Arg NII₂ IImt D-Lys Phe Arg NH₂ Hmt D-Orn Phe Arg NH₂ HmtD-Dab Phe Arg NH₂ Hmt D-Dap Phe Arg NH₂ IImt D-Arg Phe Arg NH₂ Dab =diaminobutyric Dap = diaminopropionic acid Dmt = dimethyltyrosine Mmt =2′-methyltyrosine Tmt = N, 2′,6′-trimethyltyrosine Hmt =2′-hydroxy,6′-methyltyrosine dnsDap = β-dansyl-L-α,β-diaminopropionicacid atnDap = β-anthraniloyl-L-α,β-diaminopropionic acid Bio = biotin

Examples of peptides that do not activate mu-opioid receptors include,but are not limited to, the aromatic-cationic peptides shown in Table 6.

TABLE 6 Peptide Analogs Lacking Mu-Opioid Activity Amino Amino AminoAmino Acid Acid Acid Acid C-Terminal Position 1 Position 2 Position 3Position 4 Modification D-Arg Dmt Lys Phe NH₂ D-Arg Dmt Phe Lys NH₂D-Arg Phe Lys Dmt NH₂ D-Arg Phe Dmt Lys NH₂ D-Arg Lys Dmt Phe NH₂ D-ArgLys Phc Dmt NH₂ Phe Lys Dmt D-Arg NH₂ Phe Lys D-Arg Dmt NH₂ Phe D-ArgPhe Lys NH₂ Phe D-Arg Dmt Lys NH₂ Phe D-Arg Lys Dmt NH₂ Phe Dmt D-ArgLys NH₂ Phe Dmt Lys D-Arg NH₂ Lys Phe D-Arg Dmt NH₂ Lys Phe Dmt D-ArgNH₂ Lys Dmt D-Arg Phe NH₂ Lys Dmt Phc D-Arg NH₂ Lys D-Arg Phe Dmt NH₂Lys D-Arg Dmt Phe NH₂ D-Arg Dmt D-Arg Phe NH₂ D-Arg Dmt D-Arg Dmt NII₂D-Arg Dmt D-Arg Tyr NH₂ D-Arg Dmt D-Arg Trp NH₂ Trp D-Arg Phe Lys NH₂Trp D-Arg Tyr Lys NH₂ Trp D-Arg Trp Lys NH₂ Trp D-Arg Dmt Lys NH₂ D-ArgTip Lys Phc NH₂ D-Arg Tip Phe Lys NH₂ D-Arg Tip Lys Dmt NH₂ D-Arg TipDmt Lys NH₂ D-Arg Lys Tip Phe NII₂ D-Arg Lys Tip Dmt NH₂ Cha D-Arg PheLys NH₂ Ala D-Arg Phe Lys NH₂ Cha = cyclohexylalanine

The amino acids of the peptides shown in Table 5 and 6 may be in eitherthe L- or the D-configuration.

The peptides may be synthesized by any of the methods well known in theart. Suitable methods for chemically synthesizing the protein include,for example, those described by Stuart and Young in Solid Phase PeptideSynthesis, Second Edition, Pierce Chemical Company (1984), and inMethods Enzymol., 289, Academic Press, Inc, New York (1997).

Prophylactic and Therapeutic Uses of Aromatic-Cationic Peptides.

General. The aromatic-cationic peptides described herein are useful toprevent or treat disease. Specifically, the disclosure provides for bothprophylactic and therapeutic methods of treating a subject having or atrisk of (susceptible to) heart failure. Accordingly, the present methodsprovide for the prevention and/or treatment of heart failure in asubject by administering an effective amount of an aromatic-cationicpeptide to a subject in need thereof. See Tsutsui et al. “Mitochondrialoxidative stress, DNA damage, and heart failure.” Antioxidants and RedoxSignaling. 8(9): 1737-1744 (2006).

Therapeutic Methods.

One aspect of the technology includes methods of treating heart failurein a subject for therapeutic purposes. In therapeutic applications,compositions or medicaments are administered to a subject suspected of,or already suffering from such a disease in an amount sufficient tocure, or at least partially arrest, the symptoms of the disease,including its complications and intermediate pathological phenotypes indevelopment of the disease. As such, the invention provides methods oftreating an individual afflicted with heart failure.

Subjects suffering from heart failure can be identified by any or acombination of diagnostic or prognostic assays known in the art. Forexample, typical symptoms of heart failure include shortness of breath(dyspnea), fatigue, weakness, difficulty breathing when lying flat, andswelling of the legs, ankles or abdomen (edema). The subject may also besuffering from other disorders including coronary artery disease,systemic hypertension, cardiomyopathy or myocarditis, congenital heartdisease, abnormal heart valves or valvular heart disease, severe lungdisease, diabetes, severe anemia hyperthyroidism, arrhythmia ordysrhythmia and myocardial infarction. The primary signs of congestiveheart failure are: cardiomegaly (enlarged heart), tachypnea (rapidbreathing; occurs in the case of left side failure) and hepatomegaly(enlarged liver; occurs in the case of right side failure). Acutemyocardial infarction (“AMI”) due to obstruction of a coronary artery isa common initiating event that can lead ultimately to heart failure.However, a subject that has AMI does not necessarily develop heartfailure. Likewise, subjects that suffer from heart failure did notnecessarily suffer from an AMI.

In one aspect, the present disclosure provides a method of treatinghypertensive cardiomyopathy by administering an effective amount of anaromatic-cationic peptide to a subject in need thereof. As hypertensivecardiomyopathy worsens, it can lead to congestive heart failure.Subjects suffering from hypertensive cardiomyopathy can be identified byany or a combination of diagnostic or prognostic assays known in theart. example, typical symptoms of hypertensive cardiomyopathy includehypertension (high blood pressure), cough, weakness, and fatigue.Additional symptoms of hypertensive cardiomyopathy include leg swelling,weight gain, difficulty breathing when lying flat, increasing shortnessof breath with activity, and waking in the middle of the night short ofbreath.

Prophylactic Methods.

In one aspect, the invention provides a method for preventing, in asubject, heart failure by administering to the subject anaromatic-cationic peptide that prevents the initiation or progression ofthe infection. Subjects at risk for heart failure can be identified by,e.g., any or a combination of diagnostic or prognostic assays asdescribed herein. In prophylactic applications, pharmaceuticalcompositions or medicaments of aromatic-cationic peptides areadministered to a subject susceptible to, or otherwise at risk of adisease or condition in an amount sufficient to eliminate or reduce therisk, lessen the severity, or delay the outset of the disease, includingbiochemical, histologic and/or behavioral symptoms of the disease, itscomplications and intermediate pathological phenotypes presenting duringdevelopment of the disease. Administration of a prophylacticaromatic-cationic can occur prior to the manifestation of symptomscharacteristic of the aberrancy, such that a disease or disorder isprevented or, alternatively, delayed in its progression. The appropriatecompound can be determined based on screening assays described above.

Determination of the Biological Effect of the Aromatic-CationicPeptide-Based Therapeutic.

In various embodiments, suitable in vitro or in vivo assays areperformed to determine the effect of a specific aromatic-cationicpeptide-based therapeutic and whether its administration is indicatedfor treatment. In various embodiments, in vitro assays can be performedwith representative animal models, to determine if a givenaromatic-cationic peptide-based therapeutic exerts the desired effect inpreventing or treating heart failure. Compounds for use in therapy canbe tested in suitable animal model systems including, but not limited torats, mice, chicken, cows, monkeys, rabbits, and the like, prior totesting in human subjects. Similarly, for in vivo testing, any of theanimal model system known in the art can be used prior to administrationto human subjects.

HF has been induced in different species with volume overload, pressureoverload, fast pacing, myocardial ischemia, cardiotoxic drugs, orgenetically modified models. Models using pressure overload have beenmost commonly used. Hypertension is associated with an increased riskfor the development of HF. In one mouse model, angiotensin II (Ang II)increases blood pressure and induces cardiomyocyte hypertrophy,increased cardiac fibrosis, and impaired cardiomyocyte relaxation.Infusion of angiotensin to mice by mini osmotic pump increases systolicand diastolic blood pressure, increases heart weight and leftventricular thickness (LVMI), and impaired myocardial performance index(MPI).

In a second illustrative mouse model, sustained high level expression ofGαq can lead to marked myocyte apoptosis, resulting in cardiachypertrophy and heart failure by 16 weeks of age (D'Angelo et al.,1998). The β-adrenergic receptors (βARs) are primarily coupled to theheterotrimeric G protein, Gs, to stimulate adenylyl cyclase activity.This association generates intracellular cAMP and protein kinase Aactivation, which regulate cardiac contractility and heart rate.Overexpression of Gαq leads to decreased responsiveness to β-adrenergicagonists and results in HF.

Experimental constriction of the aorta by surgical ligation is alsowidely used as a model of HF. Transaortic constriction (TAC) results inpressure overload induced HF, with increase in left ventricular (LV)mass. TAC is performed as described by Tamayski O et al. (2004) using a7-0 silk double-knot suture to constrict the ascending aorta. After TAC,mice develop HF within a period of 4 weeks.

Modes of Administration and Effective Dosages

Any method known to those in the art for contacting a cell, organ ortissue with a peptide may be employed. Suitable methods include invitro, ex vivo, or in vivo methods. In vivo methods typically includethe administration of an aromatic-cationic peptide, such as thosedescribed above, to a mammal, suitably a human. When used in vivo fortherapy, the aromatic-cationic peptides are administered to the subjectin effective amounts (i.e., amounts that have desired therapeuticeffect). The dose and dosage regimen will depend upon the degree of theinfection in the subject, the characteristics of the particulararomatic-cationic peptide used, e.g., its therapeutic index, thesubject, and the subject's history.

The effective amount may be determined during pre-clinical trials andclinical trials by methods familiar to physicians and clinicians. Aneffective amount of a peptide useful in the methods may be administeredto a mammal in need thereof by any of a number of well-known methods foradministering pharmaceutical compounds. The peptide may be administeredsystemically or locally.

The peptide may be formulated as a pharmaceutically acceptable salt. Theterm “pharmaceutically acceptable salt” means a salt prepared from abase or an acid which is acceptable for administration to a patient,such as a mammal (e.g., salts having acceptable mammalian safety for agiven dosage regime). However, it is understood that the salts are notrequired to be pharmaceutically acceptable salts, such as salts ofintermediate compounds that are not intended for administration to apatient. Pharmaceutically acceptable salts can be derived frompharmaceutically acceptable inorganic or organic bases and frompharmaceutically acceptable inorganic or organic acids. In addition,when a peptide contains both a basic moiety, such as an amine, pyridineor imidazole, and an acidic moiety such as a carboxylic acid ortetrazole, zwitterions may be formed and are included within the term“salt” as used herein. Salts derived from pharmaceutically acceptableinorganic bases include ammonium, calcium, copper, ferric, ferrous,lithium, magnesium, manganic, manganous, potassium, sodium, and zincsalts, and the like. Salts derived from pharmaceutically acceptableorganic bases include salts of primary, secondary and tertiary amines,including substituted amines, cyclic amines, naturally-occurring aminesand the like, such as arginine, betaine, caffeine, choline,N,N′-dibenzylethylenediamine, di ethylamine, 2-diethylaminoethanol,2-dimethylaminoethanol, ethanolamine, ethylenediamine,N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine,hydrabamine, isopropylamine, lysine, methylglucamine, morpholine,piperazine, piperadine, polyamine resins, procaine, purines,theobromine, triethylamine, trimethylamine, tripropylamine, tromethamineand the like. Salts derived from pharmaceutically acceptable inorganicacids include salts of boric, carbonic, hydrohalic (hydrobromic,hydrochloric, hydrofluoric or hydroiodic), nitric, phosphoric, sulfamicand sulfuric acids. Salts derived from pharmaceutically acceptableorganic acids include salts of aliphatic hydroxyl acids (e.g., citric,gluconic, glycolic, lactic, lactobionic, malic, and tartaric acids),aliphatic monocarboxylic acids (e.g., acetic, butyric, formic, propionicand trifluoroacetic acids), amino acids (e.g., aspartic and glutamicacids), aromatic carboxylic acids (e.g., benzoic, p-chlorobenzoic,diphenylacetic, gentisic, hippuric, and triphenylacetic acids), aromatichydroxyl acids (e.g., o-hydroxybenzoic, p-hydroxybenzoic,1-hydroxynaphthalene-2-carboxylic and 3-hydroxynaphthalene-2-carboxylicacids), ascorbic, dicarboxylic acids (e.g., fumaric, maleic, oxalic andsuccinic acids), glucuronic, mandelic, mucic, nicotinic, orotic, pamoic,pantothenic, sulfonic acids (e.g., benzenesulfonic, camphosulfonic,edisylic, ethanesulfonic, isethionic, methanesulfonic,naphthalenesulfonic, naphthalene-1,5-disulfonic,naphthalene-2,6-disulfonic and p-toluenesulfonic acids), xinafoic acid,and the like.

The aromatic-cationic peptides described herein can be incorporated intopharmaceutical compositions for administration, singly or incombination, to a subject for the treatment or prevention of a disorderdescribed herein. Such compositions typically include the active agentand a pharmaceutically acceptable carrier. As used herein the term“pharmaceutically acceptable carrier” includes saline, solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. Supplementary active compounds can alsobe incorporated into the compositions.

Pharmaceutical compositions are typically formulated to be compatiblewith its intended route of administration. Examples of routes ofadministration include parenteral (e.g., intravenous, intradermal,intraperitoneal or subcutaneous), oral, inhalation, transdermal(topical), intraocular, iontophoretic, and transmucosal administration.Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic. For convenience of thepatient or treating physician, the dosing formulation can be provided ina kit containing all necessary equipment (e.g., vials of drug, vials ofdiluent, syringes and needles) for a treatment course (e.g., 7 days oftreatment).

Pharmaceutical compositions suitable for injectable use can includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, a composition for parenteral administration must be sterile andshould be fluid to the extent that easy syringability exists. It shouldbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi.

The aromatic-cationic peptide compositions can include a carrier, whichcan be a solvent or dispersion medium containing, for example, water,ethanol, polyol (for example, glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Prevention of theaction of microorganisms can be achieved by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid, thiomerasol, and the like. Glutathione and otherantioxidants can be included to prevent oxidation. In many cases, itwill be preferable to include isotonic agents, for example, sugars,polyalcohols such as mannitol, sorbitol, or sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate or gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle, which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, typical methods of preparation includevacuum drying and freeze drying, which can yield a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds can be delivered in theform of an aerosol spray from a pressurized container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer. Such methods include those described in U.S. Pat. No.6,468,798.

Systemic administration of a therapeutic compound as described hereincan also be by transmucosal or transdermal means. For transmucosal ortransdermal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art, and include, for example, for transmucosaladministration, detergents, bile salts, and fusidic acid derivatives.Transmucosal administration can be accomplished through the use of nasalsprays. For transdermal administration, the active compounds areformulated into ointments, salves, gels, or creams as generally known inthe art. In one embodiment, transdermal administration may be performedmy iontophoresis.

A therapeutic protein or peptide can be formulated in a carrier system.The carrier can be a colloidal system. The colloidal system can be aliposome, a phospholipid bilayer vehicle. In one embodiment, thetherapeutic peptide is encapsulated in a liposome while maintainingpeptide integrity. As one skilled in the art would appreciate, there area variety of methods to prepare liposomes. (See Lichtenberg et al.,Methods Biochem. Anal., 33:337-462 (1988); Anselem et al., LiposomeTechnology, CRC Press (1993)). Liposomal formulations can delayclearance and increase cellular uptake (See Reddy, Ann. Pharmacother.,34(7-8):915-923 (2000)). An active agent can also be loaded into aparticle prepared from pharmaceutically acceptable ingredientsincluding, but not limited to, soluble, insoluble, permeable,impermeable, biodegradable or gastroretentive polymers or liposomes.Such particles include, but are not limited to, nanoparticles,biodegradable nanoparticles, microparticles, biodegradablemicroparticles, nanospheres, biodegradable nanospheres, microspheres,biodegradable microspheres, capsules, emulsions, liposomes, micelles andviral vector systems.

The carrier can also be a polymer, e.g., a biodegradable, biocompatiblepolymer matrix. In one embodiment, the therapeutic peptide can beembedded in the polymer matrix, while maintaining protein integrity. Thepolymer may be natural, such as polypeptides, proteins orpolysaccharides, or synthetic, such as poly α-hydroxy acids. Examplesinclude carriers made of, e.g., collagen, fibronectin, clastin,cellulose acetate, cellulose nitrate, polysaccharide, fibrin, gelatin,and combinations thereof. In one embodiment, the polymer is poly-lacticacid (PLA) or copoly lactic/glycolic acid (PGLA). The polymeric matricescan be prepared and isolated in a variety of forms and sizes, includingmicrospheres and nanospheres. Polymer formulations can lead to prolongedduration of therapeutic effect. (See Reddy, Ann. Pharmacother.,34(7-8):915-923 (2000)). A polymer formulation for human growth hormone(hGH) has been used in clinical trials. (See Kozarich and Rich, ChemicalBiology, 2:548-552 (1998)).

Examples of polymer microsphere sustained release formulations aredescribed in PCT publication WO 99/15154 (Tracy et al.), U.S. Pat. Nos.5,674,534 and 5,716,644 (both to Zale et al.), PCT publication WO96/40073 (Zale et al.), and PCT publication WO 00/38651 (Shah et al.).U.S. Pat. Nos. 5,674,534 and 5,716,644 and PCT publication WO 96/40073describe a polymeric matrix containing particles of erythropoietin thatare stabilized against aggregation with a salt.

In some embodiments, the therapeutic compounds are prepared withcarriers that will protect the therapeutic compounds against rapidelimination from the body, such as a controlled release formulation,including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoestcrs, and polylactic acid. Such formulations can be preparedusing known techniques. The materials can also be obtained commercially,e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomalsuspensions (including liposomes targeted to specific cells withmonoclonal antibodies to cell-specific antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

The therapeutic compounds can also be formulated to enhanceintracellular delivery. For example, liposomal delivery systems areknown in the art, see, e.g., Chonn and Cullis, “Recent Advances inLiposome Drug Delivery Systems,” Current Opinion in Biotechnology6:698-708 (1995); Weiner, “Liposomes for Protein Delivery: SelectingManufacture and Development Processes,” Immunomethods, 4(3):201-9(1994); and Gregoriadis, “Engineering Liposomes for Drug Delivery:Progress and Problems,” Trends Biotechnol., 13(12):527-37 (1995).Mizguchi et al., Cancer Lett., 100:63-69 (1996), describes the use offusogenic liposomes to deliver a protein to cells both in vivo and invitro.

Dosage, toxicity and therapeutic efficacy of the therapeutic agents canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD50 (the dose lethal to50% of the population) and the ED50 (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD50/ED50. Compounds which exhibit high therapeutic indices arepreferred. While compounds that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the methods, the therapeutically effective dose can be estimatedinitially from cell culture assays. A dose can be formulated in animalmodels to achieve a circulating plasma concentration range that includesthe IC50 (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma may be measured, for example, by highperformance liquid chromatography.

Typically, an effective amount of the aromatic-cationic peptides,sufficient for achieving a therapeutic or prophylactic effect, rangefrom about 0.000001 mg per kilogram body weight per day to about 10,000mg per kilogram body weight per day. Suitably, the dosage ranges arefrom about 0.0001 mg per kilogram body weight per day to about 100 mgper kilogram body weight per day. For example dosages can be 1 mg/kgbody weight or 10 mg/kg body weight every day, every two days or everythree days or within the range of 1-10 mg/kg every week, every two weeksor every three weeks. In one embodiment, a single dosage of peptideranges from 0.001-10,000 micrograms per kg body weight. In oneembodiment, aromatic-cationic peptide concentrations in a carrier rangefrom 0.2 to 2000 micrograms per delivered milliliter. An exemplarytreatment regime entails administration once per day or once a week. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and preferably until the subject shows partial orcomplete amelioration of symptoms of disease. Thereafter, the patientcan be administered a prophylactic regime.

In some embodiments, a therapeutically effective amount of anaromatic-cationic peptide may be defined as a concentration of peptideat the target tissue of 10⁻¹² to 10⁻⁶ molar, e.g., approximately 10⁻⁷molar. This concentration may be delivered by systemic doses of 0.001 to100 mg/kg or equivalent dose by body surface area. The schedule of doseswould be optimized to maintain the therapeutic concentration at thetarget tissue, most preferably by single daily or weekly administration,but also including continuous administration (e.g., parenteral infusionor transdermal application).

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to, the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of the therapeutic compositionsdescribed herein can include a single treatment or a series oftreatments.

The mammal treated in accordance present methods can be any mammal,including, for example, farm animals, such as sheep, pigs, cows, andhorses; pet animals, such as dogs and cats; laboratory animals, such asrats, mice and rabbits. In a preferred embodiment, the mammal is ahuman.

Combination Therapy with an Aromatic-Cationic Peptide and OtherTherapeutic Agents

In some embodiments, the aromatic-cationic peptides may be combined withone or more additional agents for the prevention or treatment of heartfailure. Drug treatment for heart failure typically involves diuretics,ACE inhibitors, digoxin (also called digitalis), calcium channelblockers, and beta-blockers. In mild cases, thiazide diuretics, such ashydrochlorothiazide at 25-50 mg/day or chlorothiazide at 250-500 mg/day,are useful. However, supplemental potassium chloride may be needed,since chronic diuresis causes hypokalemis alkalosis. Moreover, thiazidediuretics usually are not effective in patients with advanced symptomsof heart failure. Typical doses of ACE inhibitors include captopril at25-50 mg/day and quinapril at 10 mg/day.

In one embodiment, the aromatic-cationic peptide is combined with anadrenergic beta-2 agonist. An “adrenergic beta-2 agonist” refers toadrenergic beta-2 agonists and analogues and derivatives thereof,including, for example, natural or synthetic functional variants whichhave adrenergic beta-2 agonist biological activity, as well as fragmentsof an adrenergic beta-2 agonist having adrenergic beta-2 agonistbiological activity. The term “adrenergic beta-2 agonist biologicalactivity” refers to activity that mimics the effects of adrenaline andnoradrenaline in a subject and which improves myocardial contractilityin a patient having heart failure. Commonly known adrenergic beta-2agonists include, but are not limited to, clenbuterol, albuterol,formeoterol, levalbuterol, metaproterenol, pirbuterol, salmeterol, andterbutaline.

In one embodiment, the aromatic-cationic peptide is combined with anadrenergic beta-1 antagonist. Adrenergic beta-1 antagonists andadrenergic beta-1 blockers refer to adrenergic beta-1 antagonists andanalogues and derivatives thereof, including, for example, natural orsynthetic functional variants which have adrenergic beta-1 antagonistbiological activity, as well as fragments of an adrenergic beta-1antagonist having adrenergic beta-1 antagonist biological activity.Adrenergic beta-1 antagonist biological activity refers to activity thatblocks the effects of adrenaline on beta receptors. Commonly knownadrenergic beta-1 antagonists include, but are not limited to,acebutolol, atenolol, betaxolol, bisoprolol, esmolol, and metoprolol.

Clenbuterol, for example, is available under numerous brand namesincluding Spiropent® (Boehinger Ingelheim), Broncodil® (Von Boch I),Broncoterol® (Quimedical PT), Cesbron® (Fidelis PT), and Clenbuter®(Biomedica Foscama). Similarly, methods of preparing adrenergic beta-1antagonists such as metoprolol and their analogues and derivatives arewell-known in the art. Metoprolol, in particular, is commerciallyavailable under the brand names Lopressor® (metoprolol tartate)manufactured by Novartis Pharmaceuticals Corporation, One Health Plaza,East Hanover, N.J. 07936-1080. Generic versions of Lopressor® are alsoavailable from Mylan Laboratories Inc., 1500 Corporate Drive, Suite 400,Canonsburg, Pa. 15317; and Watson Pharmaceuticals, Inc., 360 Mt. KembleAve. Morristown, N.J. 07962. Metoprolol is also commercially availableunder the brand name Toprol XL®, manufactured by Astra Zeneca, LP.

In one embodiment, an additional therapeutic agent is administered to asubject in combination with an aromatic cationic peptide, such that asynergistic therapeutic effect is produced. A “synergistic therapeuticeffect” refers to a greater-than-additive therapeutic effect which isproduced by a combination of two therapeutic agents, and which exceedsthat which would otherwise result from individual administration ofeither therapeutic agent alone. Therefore, lower doses of one or both ofthe therapeutic agents may be used in treating heart failure, resultingin increased therapeutic efficacy and decreased side-effects.

In any case, the multiple therapeutic agents may be administered in anyorder or even simultaneously. If simultaneously, the multipletherapeutic agents may be provided in a single, unified form, or inmultiple forms (by way of example only, either as a single pill or astwo separate pills). One of the therapeutic agents may be given inmultiple doses, or both may be given as multiple doses. If notsimultaneous, the timing between the multiple doses may vary from morethan zero weeks to less than four weeks. In addition, the combinationmethods, compositions and formulations are not to be limited to the useof only two agents.

EXAMPLES

The present invention is further illustrated by the following examples,which should not be construed as limiting in any way.

Example 1 Effects of Aromatic-Cationic Peptides in Mouse Models of HeartFailure

In this Example, the effect of reducing mitochondrial oxidative stressby the mitochondrial-targeted antioxidant peptide (SS-31) inhypertensive cardiomyopathy and heart failure was investigated. Themitochondrial targeted antioxidant peptide SS-31 was used to determinethe role of NADPH oxidase and mitochondria in Angiotensin II (AngII)-induced cardiomyopathy, as well as in Gαq overcxpressing mice withheart failure.

Methods

Neonatal Mouse Cardiomyocyte Culture and Flow Cytometry.

Ventricles from mouse neonates younger than 72 hours were dissected,minced, and enzymatically digested with Blendzyme 4 (45 μg/ml, Roche).After enzymatic digestion, cardiomyocytes were enriched usingdifferential pre-plating for 2 hours, then seeded on fibronectin-coatedculture dishes for 24 hours in DMEM (Gibco), with 20% Fetal Bovine Serum(Sigma) and 25 μM Arabinosylcytosine (Sigma). Cardiomyocytes werestimulated with Angiotensin II (1 μM) for 3 hours in serum-free DMEMcontaining 0.5% insulin transferrin-selenium (Sigma), 2 mM glutamine,and 1 mg/ml of BSA. Cardiomyocytes were simultaneously treated witheither of the following: SS-31 (1 nM), N-acetyl cysteine (NAC: 0.5 mM),or PBS control. To measure mitochondrial superoxide concentration,Mitosox (5 μM) was incubated for 30 min at 37° C. to loadcardiomyocytes, followed by 2 washes, with Hanks Balanced Salt Solution.Samples were analyzed using excitation/emission of 488/625 nm by flowcytometry. Flow data was analyzed using FCS Express (De Novo Software,Los Angeles, Calif.), and presented as histogram distributions ofMitosox fluorescence intensity.

Mouse Experiments, Drug Delivery, Echocardiography and Blood PressureMeasurement.

All animal experiments were approved by the University of WashingtonInstitutional Animal Care and Use Committee. C57BL6 mice were housed ina barrier specific pathogen-free facility. Six to ten mice were includedin each experimental group (Saline, Ang II, Ang II+SS-31, WT, Gαq,Gαq+SS-31). A pressor dose of Ang II (1.1 mg/kg/d) was continuouslyadministered for 4 weeks using subcutaneous Alzet 1004 osmoticminipumps, with or without addition of SS-31 (3 mg/kg/d).Echocardiography was performed at baseline and after 4 weeks of pumpimplantation using a Siemens Acuson CV-70 equipped with a 13 MHz probe.Under 0.5% isoflurane to reduce agitation, standard M-mode, conventionaland Tissue Doppler images were taken, and functional calculations wereperformed according to American Society of Echocardiography guidelines.MPI was calculated as the ratio of the sum of isovolemic contraction andrelaxation time to LV ejection time. An increase in MPI is an indicationthat a greater fraction of systole is spent to cope with the pressurechanges during the isovolemic phases. As a reference for SS-31 peptideeffect in Ang II treated mice, a genetic mouse model of Rosa-26inducible-mCAT was included, in which mitochondrial catalase wasoverexpressed two weeks before Ang II treatment.

Blood pressure was measured in a separate group of mice by telemetryusing an intravascular catheter PA-C10 (DSI, MN), in which measurementwas performed every three hours starting from 2 days before pumpplacement until 2 days after Ang pump placement. After this time, a newpump loaded with Ang II+SS-31 was inserted, followed by another 2 daysof recording to see if SS-31 had an effect on blood pressure.

Quantitative Pathology.

Ventricular tissues were cut into transverse slices, and subsequentlyembedded with paraffin, sectioned, and subjected to Masson Trichromestaining. Quantitative analysis of fibrosis was performed by measuringthe percentage of blue-staining fibrotic tissue relative to the totalcross-sectional area of the ventricles.

Measurement of Mitochondrial Protein Carbonyl Groups.

For mitochondrial protein extraction, ventricular tissues werehomogenized in mitochondrial isolation buffer (1 mM EGTA, 10 mM HEPES,250 mM sucrose, 10 mM Tris-HCl, pH 7.4). The lysates were centrifugedfor 7 min at 800 g in 4° C. The supernatants were then centrifuged for30 min at 4000 g in 4° C. The crude mitochondria pellets wereresuspended in small volume of mitochondrial isolation buffer, sonicatedon ice to disrupt the membrane, and treated with 1% streptomycin sulfateto precipitate mitochondrial nucleic acids. The OxiSelect™ ProteinCarbonyl ELISA Kit (Cell Biolabs) was used to analyze 1 μg of proteinsample per assay. The ELISA was performed according to the instructionmanual, with slight modification. Briefly, protein samples were reactedwith dinitrophenylhydrazine (DNPH) and probed with anti-DNPH antibody,followed by HRP conjugated secondary antibody. The anti-DNPH antibodyand HRP conjugated secondary antibody concentrations were 1:2500 and1:4000, respectively.

Quantitative PCR.

Gene expression was quantified by quantitative real time PCR using anApplied Biosystems 7900 themocycler with Taqman Gene Expression Assayson Demand, which included: PGC1-α (Mm00731216), TFAM (Mm00447485), NRF-1(Mm00447996), NRF-2 (Mm00487471), Collagen 1a2 (Mm00483937), and ANP(Mm01255747). Expression assays were normalized to 18S RNA.

NADPH Oxidase Activity.

The NADPH oxidase assay was performed as described elsewhere. In brief,10 μg of ventricular protein extract was incubated with dihydroethidium(DHE, 10 μM), sperm DNA (1.25 μg/ml), and NADPH (50 μM) in PBS/DTPA(containing 100 μM DTPA), The assay was incubated at 37° C. in the darkfor 30 min and the fluorescence was detected using excitation/emissionof 490/580 nm.

Western Immunoblots.

Cardiac protein extracts were prepared by homogenization in lysis buffercontaining protease and phosphatase inhibitors on ice (1.5 mM KCl, 50 mMTris HCl, 0.125% Sodium deoxycholate, 0.375% Triton× 100, 0.15% NP40, 3mM EDTA). The samples were sonicated and centrifuged at 10,000 g for 15min in 4° C. The supernatant was collected and the concentration wasdetermined using a BCA assay (Pierce Thermo Scientific, Rockford, Ill.).Total protein (25 μg) was separated on NuPAGE 4-12% Bis-Tris gel(Invitrogen) and transferred to 0.45 μm PVDF membrane (Millipore), andthen blocked in 5% non-fat dry milk in Tris-buffer solution with 0.1%Tween-20 for 1 hour. Primary antibodies were incubated overnight, andsecondary antibodies were incubated for 1 hour. The primary antibodiesincluded: rabbit monoclonal anti-cleaved caspase-3 (Cell Signaling),mouse monoclonal anti-GAPDH (Millipore), rabbit polyclonal phospho-p38MAP kinasc (Cell Signaling), and mouse monoclonal anti-p38 (Santa CruzBiotechnology). The enhanced chemiluminescence method (ThermoScientific) was used for detection. Image Quant ver.2.0 was used toquantified the relative band density as a ratio to GAPDH (internalcontrol). All samples were normalized to the same cardiac proteinsample.

Statistical Analysis.

All data are presented as mean±SEM. Comparisons between two groups areperformed using Student t-tests. One-way ANOVA was used to comparedifferences among multiple groups, followed by Tukey post-hoc test forsignificance. P<0.05 were considered significant.

Results

Ang-II Increased Mitochondrial ROS in Neonatal Cardiomyoctyes, which wasAlleviated by Mitochondrial Antioxidant Peptide SS-31.

Flow cytometry analysis demonstrated that Angiotensin TI increasedMitosox fluorescence (an indicator of mitochondrial superoxide) byapproximately 2-fold in neonatal cardiomyocytes. Treatment with N-acetylcysteine (NAC), a non-targeted antioxidant drug, did not show any effecton the level of mitochondrial ROS after Ang II. In contrast, SS-31reduced Ang II-induced mitosox fluorescence to the level similar to thatof saline treated cardiomyocytes (FIG. 1). These results indicated thatAng II induced mitochondrial oxidative stress in cardiomyocytes thatcould only be alleviated by a mitochondrial targeted antioxidant.

SS-31 Peptide Ameliorates Ang-II Induced Cardiomyopathy Despite theAbsence of Blood Pressure Lowering Effect.

To recapitulate hypertensive cardiomyopathy, a pressor dose of Ang II(1.1 mg/kg/d) was administered for 4 weeks via subcutaneous continuousdelivery with Alzet 1004 osmotic minipumps. As shown in FIGS. 2A and 2B,intravascular telemetry revealed that this dose of Ang II significantlyincreased systolic and diastolic blood pressure by 25-28 mm Hg abovebaseline (BP: 118.8±4.0/94.5±3.5 mm Hg at baseline vs.146.0±5.6/119.3±4.0 mm Hg after Ang II, p<0.001). Simultaneousadministration of SS-31 (3 mg/kg/d) did not show any effect on bloodpressure (FIG. 2).

After 4 weeks of Ang II, echocardiography revealed an approximately2-fold increase in left ventricular mass index (LVMI) compared withbaseline (FIG. 3A), no change in LV end diastolic diameter (LVEDD, FIG.3B) or systolic function as measured by fractional shortening (FS, FIG.3C), and ˜35% decline in Ea/Aa, an indicator of diastolic function (FIG.3D). Simultaneous administration of SS-31 significantly ameliorated AngII induced cardiac hypertrophy and diastolic dysfunction, with 33%reduction of LVMI (Ang: 6.32±0.39 mg/g vs. Ang+SS-31: 4.21±0.17 mg/g,p=0.001, FIG. 3A left panel) and 38% better preservation of Ea/Aa (Ang:0.723±0.15 vs. Ang+SS-31: 1.17±0.11, p=0.04, FIG. 3D left panel). Theseeffects were comparable to the beneficial effects of catalase targetedto mitochondria (i-mCAT), in which induction of mitochondrial catalasetwo weeks before Ang II treatment conferred protection against Ang IIinduced cardiac hypertrophy and diastolic dysfunction (FIG. 3A-D, rightpanels). FIG. 4A demonstrated that Ang II increased heart weights by 45%above those of saline treated control hearts (5.3±0.18 in saline vs.7.69±0.20 in Ang, p<0.001) and SS-31 reduced heart weights to 6.05±0.135mg/mm (p<0.01 compared with Ang alone). The phenotype of cardiachypertrophy was confirmed by quantitative PCR for atrial natriureticpeptide (ANP), a fetal gene known to be reactivated during hypertrophy.Ang II induced ˜15 fold increased ANP gene expression, and this wasalmost completely protected by SS-31 (FIG. 4B).

The cardiac pathology was examined by Masson trichrome staining, whichdemonstrated perivascular fibrosis and interstitial fibrosis after 4weeks of Ang II (FIG. 4C). Quantitative image analysis of ventricularfibrosis (blue staining on trichrome) showed that Ang II significantlyincrease ventricular fibrosis by greater than 3 fold, which was fullyattenuated by SS-31 (FIG. 4D). The increase in cardiac fibrosis wasconfirmed by quantitative PCR of the procollagen 1a2 gene, the maincomponent of fibrosis. As shown in FIG. 4E, Ang II increased Coll1a2gene expression by ˜2.5 fold, which was almost fully attenuated by SS-31administration.

Ang II Induced Mitochondrial Protein Oxidative Damage and Signaling forMitochondrial Biogenesis.

Consistent with the finding that Ang II induced mitochondrial ROS incardiomyocytes (FIG. 1), chronic administration of Ang II for 4 weekssignificantly increased ventricular mitochondrial protein carbonylcontent, which is an indicator of protein oxidative damage (p=0.03, FIG.5A). Mitochondrial targeted antioxidant SS-31 significantly reducedcardiac mitochondrial protein carbonyls (p=0.02, FIG. 5A).

Peroxisome proliferator-activated receptor gamma co-activator (PGC-1α)has been shown to be a master regulator of mitochondrial biogenesis,regulating nuclear respiratory factors (NRFs) and mitochondrialtranscription factor A (TFAM), which transcribe nuclear DNA andmitochondrial DNA encoded mitochondrial proteins, respectively. As ROSinduced mitochondrial damage has been shown to upregulate signaling formitochondrial biogenesis, it was demonstrated that Ang II also inducedthe expression of PGC-1α and its downstream target genes, includingTFAM, NRF-1 and NRF-2 (FIG. 5B). The mitochondrial antioxidant SS-31 wasfound to fully prevent the upregulation of PGC-1α and all the downstreamtarget genes after 4 weeks of Ang II (p<0.05 for all, FIG. 5B).

SS-31 Acts Downstream of NADPH Oxidase and Reduces Activation of p38MAPK and Apoptosis in Response to Ang II.

Consistent with previous reports, 4 weeks of Ang II significantlyincreased cardiac NADPH oxidase activity (p=0.03, FIG. 6A), however,this was not changed by SS-31 administration (p=0.67, FIG. 6A),suggesting that SS-31 protection acts downstream of NADPH oxidase.

Ang II has been shown to activate several mitogen activated proteinkinase (MAPK), such as p38. It was confirmed that Ang for 4 weeksincreased phosphorylation of p38 MAPK, and this phosphorylation wassignificantly and nearly fully attenuated by SS-31 (FIG. 6C), suggestingthat this MAP kinase is activated through mt-ROS sensitive mechanisms.Mitochondrial ROS, either directly, or indirectly by activatingapoptosis signal-regulating kinase, may induce apoptosis. It was foundthat Ang II did indeed induce cardiac apoptosis, as shown by anapproximately 3-fold increase in cleaved (activated) caspase-3 in leftventricular tissue (p=0.006, FIG. 6B). SS-31 completely prevented theactivation of caspase-3 caused by Ang II (p=0.004, FIG. 6B).

SS-31 Partially Rescued Gαq Overexpression-Induced Heart Failure.

Gαq protein is coupled to receptors for catecholamines and Ang II, allof which are known to be key mediators in hypertensive cardiovasculardiseases. To extend these observations to a model of chroniccatecholamine/Ang II stimulation, a genetic mouse model with cardiacspecific overexpression of Gαq was used, which causes heart failure inmice by 14-16 weeks of age. The Gαq mice in this study had impairment ofsystolic function at 16 weeks age, as shown by a substantial decline inFS (FIG. 7A), with enlargement of the LV chamber (FIG. 7B), impairmentof diastolic function indicated by decreased Ea/Aa (FIG. 7C) andworsening of myocardial performance index (MPI, FIG. 7D). SS-31administered from 12 to 16 weeks of age (3 mg/kg/d) significantlyameliorated systolic function (p<0.001 vs. untreated Gαq, FIG. 7A) andimproved myocardial performance (p=0.04, FIG. 7D). LV chamberenlargement was slightly reduced (p=0.08, FIG. 7B), and Ea/Aa was betterpreserved by SS-31 with borderline significance (p=0.06, FIG. 7C). At 16weeks of age, normalized heart weights of Gαq mice increased by 33%,while SS-31 substantially reduced cardiac enlargement (p=0.001, FIG.7E). Lung weights significantly increased by 22% in Gαq mice, indicatinglung congestion, and this was slightly attenuated by SS-31 withborderline significance (p=0.09, FIG. 7E). Ventricular fibrosisincreased by approximately 2 fold in Gαq mice, which was not changed inSS-31 treated mice (FIG. 9A), and this was confirmed by procollagen 1a2quantitative PCR (FIG. 9B). Mitochondrial protein oxidative damage isalso evident in Gαq hearts (p=0.01, FIG. 9C), and SS-31 treated micedisplayed significant reduction of cardiac mitochondrial proteincarbonyls (p=0.05, FIG. 9C). There was no evidence of increasedcleaved-caspase 3 in Gαq mouse hearts (data not shown).

Discussion

The current study demonstrated that exposure to Ang II for 4 weeksincreased cardiac mitochondrial protein oxidative damage and induced thesignaling for mitochondrial biogenesis (FIG. 5), consistent with theprevious report that H₂O₂ directly activates transcription of PGC-1α,the master regulator of mitochondrial biogenesis. SS-31 significantlyattenuated Ang-induced mitochondrial oxidative stress and hence reducedupregulation of mitochondrial biogenesis, as well as reduced ROSmediated signaling, such as phosphorylation of p38 MAPK (FIG. 8).Furthermore, mitochondrial oxidative stress can lead to apoptosis as aresult of cytochrome c release and activation of procaspase-9, followedby caspase-3 activation and apoptosis. These results confirm thatattenuation of mitochondrial ROS with SS-31 prevented apoptosis asmeasured by activated caspase-3 (FIG. 6), concomitant with ameliorationof Ang II induced cardiac hypertrophy, fibrosis and diastolicdysfunction (FIGS. 3 and 4).

To recapitulate the effect of prolonged neurohormonal stimulation asseen in chronic hypertension on the heart, transgenic miceoverexpressing the Gαq protein were used. Gαq is a subunit of theG-protein that is coupled to adrenergic and angiotensin II receptors.Cardiac-specific overexpression of Gαq has been shown to cause heartfailure in mice by 14-16 weeks of age, despite the absence of increasedblood pressure. Twelve-week-old Gαq mice were treated with SS-31 for 4weeks and demonstrated that SS-31 partially rescued the heart failurephenotype in the Gαq mouse model. SS-31 significantly amelioratedsystolic dysfunction, cardiac hypertrophy, and improved overallmyocardial performance (FIG. 7A, D, E). A trend showing that SS-31attenuated chamber enlargement, diastolic dysfunction and lungcongestion was also observed (FIGS. 7B, C and E).

Hypertension is a highly prevalent disease that imposes a major risk forthe development of atherosclerosis, cardiomyopathy, stroke, suddencardiac death and heart failure. Hypertension-induced heart failure maybe manifested as systolic heart failure or heart failure with preservedejection fraction (HFpEF); the latter accounts for nearly half of thepatients with heart failure, especially among female elderly patients,and the prognosis of HFpEF is marginally better than that of systolicheart failure. Several clinical trials have shown that the currentrecommended antihypertensive medications are effective in reducing majorcardiovascular events and the development of heart failure by only up to50%. Although this treatment reduces mortality and improves quality oflife in patients with established systolic heart failure, there is noconvincing evidence for any effective treatment of HFpEF. Thisunderscores the urgent need to develop new prevention and treatmentstrategies for hypertensive cardiovascular diseases.

In summary, these results indicate that the mitochondrial targetedantioxidant SS-31 is beneficial in amelioration of cardiomyopathyresulting from prolonged Ang II stimulation as well as Gαqoverexpression, suggesting its potential clinical application for targetorgan protection in hypertensive cardiovascular diseases. As such, thearomatic-cationic peptides of the invention are useful in methods fortreating or preventing HF in mammalian subjects.

Example 2 Treatment or Prevention of Heart Failure in an Animal ModelUsing SS-20 (Prophetic)

The effects of the aromatic-cationic peptide SS-20 in treating orpreventing heart failure are examined in an Ang II mouse model or a Gαqmouse model.

The study is carried out in C57B16 mice. Ang II is infused by osmoticminipump (4 weeks @ 1.1 mg/kg/d) to the mice in the following groups:(1) wild type (WT); (2) transgenic mice exhibiting cardiac specificoverexpression of angiotensinogen (Tg); (3) mice overexpressing catalasetargeted to peroxisomes (pCAT); mice overexpressing catalase targetingto mitochondria (mCAT); and (4) mice expressing an inducible mCAT(i-mCAT). In mice that receive SS-20, the SS-20 is put into the sameminipump with Ang II and infused at a rate of 3 mg/kg/d for 4 weeks.

Alternatively, the study is carried out in C57B16 mice in the followingtreatment groups: (1) WT (wild-type C57B16 mice); (2) pCAT(overexpression of catalase targeted to peroxisomes); (3) mCAT(overexpression of catalase targeted to mitochondria); (4) Gαq(overexpression of Gαaq); (5) Gαq/mCAT (overexpression of Gαq andmitochondria-targeted catalase); (6) Gαq/pCAT (overexpression of Gαq andperoxisome-targeted catalase); and (7) Gαq+SS-peptides (Gαq mice treatedwith SS-20).

Cardiac function is determined by echocardiography (Acuson CV-70,Siemens Medical Systems, Malvern, Pa.) using standard imagingplanes—M-mode, conventional, and tissue Doppler imaging. The myocardialperformance index (MPI), left ventricular mass index (LVMI), and Ea/Aaratio are measured as described in Example 1.

Treatment with SS-20 is predicted to reduce Ang II-induced HF orGαq-induced HF as evidenced by a reduction in LVMI and MPT, and noincrease in heart weight. There is also predicted to be an improvementin both systolic and diastolic function. The protection provided bySS-20 is expected to be similar to the protection provided byoverexpression of catalase in mitochondria. As such, aromatic-cationicpeptide SS-20 is useful in methods at preventing or treatinghypertensive cardiomyopathy and HF in mammalian subjects.

EQUIVALENTS

The present invention is not to be limited in terms of the particularembodiments described in this application, which are intended as singleillustrations of individual aspects of the invention. Many modificationsand variations of this invention can be made without departing from itsspirit and scope, as will be apparent to those skilled in the art.Functionally equivalent methods and apparatuses within the scope of theinvention, in addition to those enumerated herein, will be apparent tothose skilled in the art from the foregoing descriptions. Suchmodifications and variations are intended to fall within the scope ofthe appended claims. The present invention is to be limited only by theterms of the appended claims, along with the full scope of equivalentsto which such claims are entitled. It is to be understood that thisinvention is not limited to particular methods, reagents, compoundscompositions or biological systems, which can, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the like,include the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 cells refers to groupshaving 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers togroups having 1, 2, 3, 4, or 5 cells, and so forth.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Other embodiments are set forth within the following claims.

What is claimed is:
 1. A method of treating heart failure orhypertensive cardiomyopathy in a mammalian subject, comprisingadministering to the mammalian subject in need thereof a therapeuticallyeffective amount of the peptide D-Arg-2′6′-Dmt-Lys-Phe-NH₂ orPhe-D-Arg-Phe-Lys-NH₂.
 2. The method of claim 1, wherein the peptide isD-Arg-2′6′-Dmt-Lys-Phe-NH₂.
 3. The method of claim 1, wherein thepeptide is Phe-D-Arg-Phe-Lys-NH₂.
 4. The method of claim 1, wherein thesubject is suffering from heart failure.
 5. The method of claim 4,wherein the heart failure results from hypertension; ischemic heartdisease; exposure to a cardiotoxic compound; myocarditis; thyroiddisease; viral infection; gingivitis; drug abuse; alcohol abuse;pericarditis; atherosclerosis; vascular disease; hypertrophiccardiomyopathy; acute myocardial infarction; left ventricular systolicdysfunction; coronary bypass surgery; starvation; an eating disorder; ora genetic defect.
 6. The method of claim 1, wherein the subject issuffering hypertensive cardiomyopathy.
 7. The method of claim 1, whereinmyocardial contractility and cardiac output in the subject administeredthe peptide are increased compared to a control subject not administeredthe peptide.
 8. The method of claim 7, wherein the myocardialcontractility and cardiac output in the subject are increased at least10% compared to a control subject not administered the peptide.
 9. Themethod of claim 1, wherein the subject is a human.
 10. The method ofclaim 1, wherein the peptide is administered orally, topically,systemically, intravenously, subcutaneously, intraperitoneally, orintramuscularly.
 11. The method of claim 1, further comprisingseparately, sequentially or simultaneously administering acardiovascular agent to the subject.
 12. The method of claim 11, whereinthe cardiovascular agent is selected from the group consisting of: ananti-arrhthymia agent, a vasodilator, an anti-anginal agent, acorticosteroid, a cardioglycoside, a diuretic, a sedative, anangiotensin converting enzyme (ACE) inhibitor, an angiotensin IIantagonist, a thrombolytic agent, a calcium channel blocker, athroboxane receptor antagonist, a radical scavenger, an anti-plateletdrug, a β-adrenaline receptor blocking drug, α-receptor blocking drug, asympathetic nerve inhibitor, a digitalis formulation, an inotrope, andan antihyperlipidemic drug.
 13. A method for increasing myocardialcontractility and cardiac output in a subject suffering from heartfailure or hypertensive cardiomyopathy comprising administering to thesubject a therapeutically effective amount of the peptideD-Arg-2′6′-Dmt-Lys-Phe-NH₂ or Phe-D-Arg-Phe-Lys-NH₂.